Acute cardiac allograft cellular rejection remains a significant source of morbidity and mortality within the first year after heart transplantation. Afterwards, cardiac allograft vasculopathy (CAV), as a result of chronic vascular rejection, is the major cause of morbidity and mortality. Within the first year post-transplantation, almost two-thirds of recipients will experience at least one rejection episode. At five years post-transplantation, nearly 50% of survivors will have CAV. In the first year after transplantation, nearly 63% of patients experience at least one episode of cardiac rejection and approximately one third of these patients will have multiple episodes. The clinical symptoms of acute cardiac rejection are relatively nonspecific (fatigue, dyspnea, low grade fever). Most CAV patients remain asymptomatic until they develop serious problems such as myocardial infarction, heart failure, ventricular dysrhythmias or sudden cardiac death. No widely accepted noninvasive method exists for the accurate diagnosis of acute or chronic cardiac rejection. Noninvasive methods such as electrocardiography, echocardiography, and nuclear studies all have been studied, but have been unsuccessful, thus far, for either condition. Several methodologies have been studied including electrocardiography, echocardiography, nuclear imaging, and phosphorus spectroscopy without success. The current gold standard for the diagnosis of acute cellular rejection remains right ventricular endomyocardial biopsy. This is an invasive method of diagnosis subject to morbidity and random sampling and interpretation error. Likewise, the gold standard for diagnosing CAV is cardiac catheterization with intravascular ultrasound, an invasive procedure also subject to morbidity. We are applying functional genomics to study acute cardiac allograft cellular rejection and CAV. Our group has developed and tested standard laboratory procedures for sample processing and if necessary amplification. We have established laboratory and bioinformatics infrastructure to support oligonucleotide microarray investigations. Two major local transplant programs (Johns Hopkins University and INOVA-Fairfax) have been or are currently collaborating. We have an IRB approved protocol which as enrolled 187 individuals to date. We hypothesize that large scale expression profiling of circulating peripheral blood mononuclear cells (PBMC, predominantly T lymphocytes) will identify genes that can serve as reliable biomarkers of acute and chronic cardiac cellular rejection. In the initial bench phase of the project, PBMCs are obtained from heart transplant recipients during periods of immunological tolerance of the allograft (no acute rejection) and immunologic intolerance of the allograft (acute rejection) and from heart transplant recipients with and without CAV to determine whether unique gene expression patterns are associated with each state. Other analytic tools that may be employed include proteomics, RT-PCR, Western blot, insitu-hybridization, immunohistochemistry, and histopathology. In the latter phase of the project we hope to translate these profiles into an acceptable bedside test for acute and chronic cardiac allograft cellular rejection. In addition to developing a biomarker approach to the diagnosis of rejection in cardiac transplant patients, expression profiling has the potential to identify immunoregulatory pathways that can serve as new targets for immunosuppressive therapy (rational drug development). In 2010, we processed 64 samples for high density oligonucleotide microarray analysis. In the latter part of 2010, a second batch containing 168 samples was processed to TRNA, the required step before going to microarrays. In 2011, an additional 139 samples were processed to TRNA and 89 samples were further processed for high density oligonucleotide microarray analysis. In October 2011 through AAugust 2012 reporting period we processed 44 samples to TRNA and processed 397 TRNA samples to a RNA stable state. In the 2012 through 2013 reporting period we processed an additional 30 samples to TRNA. In the 2013 through 2014 reporting period we obtained an additional 17 blood samples temporally related to 17 heart biopsies and processed 64 prior samples to TRNA. In 2014 through 2015, we obtained 15 blood samples temporally related to 15 heart biopsies. There are still 4 enrolled subjects who have not been transplanted. Two are inactive and two are awaiting transplant. In addition, we plan to enroll healthy volunteers to match our transplant subjects already enrolled. Recently, several published reports have established that detection of donor DNA in recipients blood can serve as a diagnostic tool of graft injury. The level of donor DNA measured as percentage of circulating cell-free donor DNA (%ccfdDNA) accurately diagnoses acute rejection with a high sensitivity and specificity, at times several months before the diagnosis by examining endomyocardial biopsies. The ability of cell free DNA to diagnose graft injury early opens a new window to re-examine markers of rejection. These markers are traditionally evaluated using biopsy results, often positive late during rejection. %ccfdDNA offers an opportunity to better characterize our analyses. In collaboration with the NHLBI laboratory of Transplantation Genomics under the direction of Dr. Hannah Valentine, we are embarking on biomarker discovery using our collected samples and their recently developed genomic approaches. Peptidomics has also emerged as a viable and promising diagnostic tool and has been applied to diseases such as tuberculosis and chemotherapy-induced heart disease. Using this tool, Dr. Hus group at The Houston Methodist Hospital Research Institute (THMHRI) in Houston, Texas has identified peptide biomarkers of diagnostic and prognostic significance in these disease entities. During this reporting period we have embarked on a collaborative biomarker discovery study with Dr. Hu focusing on using peptidomics and exosomes. The goal is to identify markers of graft injury as well as markers that can identify the cause of injury. Our current study has created at the NIH a bio-bank of samples from transplant patients with well-characterized clinical phenotypes . Under a newly executed MTA we are currently in the process of sharing a subset of our plasma samples from our bio-bank with Dr. Tony Hu of THMHRI for peptidomic and exosomic biomarker discovery.